Anti CEACAM1 antibodies and methods of using same

ABSTRACT

A hybridoma cell which has been deposited under ATCC Accession Number PTA-9974 is disclosed. Also provided are Antibodies and methods of using same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 National Phase Entry Applicationfrom PCT/IL2010/000348, filed Apr. 29, 2010, and designating the UnitedStates, which claims the benefit of U.S. Provisional Application No.61/213,040, filed on Apr. 30, 2009, which are incorporated herein intheir entireties.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates toanti-CEACAM1 antibodies, hybridoma cells producing same and methods ofusing same.

The transmembrane protein CEACAM1 [also known as biliary glycoprotein(BGP), CD66a and C-CAM1] is a member of the carcinoembryonic antigenfamily (CEA) that also belongs to the immunoglobulin superfamily.CEACAM1 interacts with other known CD66 proteins, including CD66a,CD66c, and CD66e proteins. It is expressed on a wide spectrum of cells,ranging from epithelial cells to those of hemopoietic origin (e.g.immune cells).

Many different functions have been attributed to the CEACAM1 protein. Itwas shown that the CEACAM1 protein exhibits anti-proliferativeproperties in carcinomas of colon, prostate, as well as other types ofcancer. Additional data support the central involvement of CEACAM1 inangiogenesis and metastasis. CEACAM1 also plays a role in the modulationof innate and adaptive immune responses. For example, CEACAM1 was shownto be an inhibitory receptor for activated T cells contained within thehuman intestinal epithelium [see WO99/52552 and Morales et al. J.Immunol. 163 (1999), 1363-1370]. Additional reports have indicated thatCEACAM1 engagement either by TCR cross-linking with mAb or by Neisseriagonorrhoeae Opa proteins inhibits T cell activation and proliferation.

Melanoma is a malignancy of pigment-producing cells (melanocytes),responsible for 75% of skin cancer-related incidence worldwide, mainlydue to extensive metastasis. Metastatic melanoma (MM) responds feebly tomost anticancer regimens and overall survival mean for patients with MMis 8.5 months. CEACAM1 is rarely expressed by normal melanocytes, butfrequently found on melanoma cells. CEACAM1 expression on primarycutaneous melanoma lesions strongly predicts the development ofmetastatic disease with poor prognosis. Moreover, increased CEACAM1expression was observed on NK cells derived from some patients withmetastatic melanoma compared with healthy donors.

WO2007/063424 and U.S. Patent Application No. 20070110668 disclosemethods for regulating the immune system, and in particular methods forthe regulation of a specific immune response, including the regulationof lymphocyte activity. These methods comprise both the negative andpositive modulation of CEACAM1 protein function.

U.S. Patent Application No. 20070071758 discloses methods andcompositions for the treatment and diagnosis of cancers. Specifically,U.S. Patent Application No. 20070071758 teaches methods and compositionsfor enhancing the efficacy of tumor-infiltrating lymphocyte (TIL)therapy in the treatment of cancer by negatively modulating the activityof the CEACAM1 protein, such as for example, by using an immunoglobulinspecific for CEACAM1.

U.S. Patent Application No. 20080108140 discloses methods of modulatingspecific immune responses to create a protective immunity in thetreatment of autoimmune diseases and diseases requiring thetransplantation of tissue. In particular, U.S. Patent Application No.20080108140 relates to the suppression of immune responses in a targetedfashion, by increasing the functional concentration of the CEACAM1protein in the target tissue.

U.S. Patent Application No. 20040047858 discloses specific antibodies(i.e. 34B1, 26H7 and 5F4) which are capable of modulating T cellactivity via CEACAM1 and uses thereof such as in treating immuneresponse related diseases (e.g. graft versus host disease, autoimmunediseases, cancers etc.).

U.S. Patent Application Nos. 20020028203, 20050169922 and 20080102071disclose compositions which bind T cell inhibitory receptor moleculesand modulate (i.e. enhance or suppress) T cell activity (e.g.cytotoxicity and proliferation), such as biliary glycoprotein bindingagents, and methods of using such compositions such as for treatment ofdiseases (e.g. an autoimmune disease, immunodeficiency, cancer etc.).

Other related art:

5F4 mAb: Regulation of human intestinal intraepithelial lymphocytecytolytic function by biliary glycoprotein (CD66a) [Morales V M et al.,J Immunol. (1999) 163(3): 1363-70].

GM8G5 and 29H2—both available commercially from Abcam Inc.abcamdotcomdotportal.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a hybridoma cell which has been deposited under ATCCAccession Number PTA-9974.

According to an aspect of some embodiments of the present inventionthere is provided an isolated antibody or antibody fragment comprisingan antigen recognition domain having the CDR sequences and orientationof the antibody produced from the hybridoma cell.

According to an aspect of some embodiments of the present inventionthere is provided a method of immunomodulation, the method comprisingcontacting a CEACAM1-expressing lymphocyte with the antibody or antibodyfragment.

According to an aspect of some embodiments of the present inventionthere is provided a method of inhibiting migration or proliferation of aCEACAM1 expressing tumor cell, the method comprising contacting theCEACAM1 expressing tumor cell with the antibody or antibody fragment,thereby inhibiting migration or proliferation of a CEACAM1 expressingtumor cell.

According to an aspect of some embodiments of the present inventionthere is provided a method for diagnosing a cancer in a subject in needthereof, the method comprising contacting a biological sample derivedfrom the subject with the antibody or antibody fragment, wherein acomplex formation beyond a predetermined threshold is indicative of thecancer in the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the antibody or antibody fragment, thereby treating the cancerin the subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of inhibiting CEACAM1 homotypic orheterotypic protein-protein interaction, the method comprisingcontacting a CEACAM1-expressing lymphocyte with the antibody or antibodyfragment, thereby inhibiting CEACAM1 homotypic or heterotypicprotein-protein interaction.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as an activeingredient the antibody or antibody fragment.

According to some embodiments of the invention, the isolated antibody orantibody fragment is attached to a cytotoxic moiety.

According to some embodiments of the invention, the cytotoxic moietycomprises a cytotoxin, a chemokine, a chemotherapy, a pro-apoptotic, aninterferon, a radioactive moiety, or combinations thereof.

According to some embodiments of the invention, the isolated antibody orantibody fragment is attached to an identifiable moiety.

According to some embodiments of the invention, cells of the cancer arecharacterized by over expression of CEACAM1 as compared to unaffectedcells.

According to some embodiments of the invention, the method of treatingcancer further comprises administering to the subject lymphocytes.

According to some embodiments of the invention, the lymphocytes compriseT cells or NK cells.

According to some embodiments of the invention, the CEACAM1-expressinglymphocyte is a Tumor Infiltrating Lymphocyte or NK cell.

According to some embodiments of the invention, the CEACAM1-expressinglymphocyte is a cytotoxic T cell.

According to some embodiments of the invention, the tumor cell comprisesa melanoma tumor cell.

According to some embodiments of the invention, the cancer is melanoma.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-B depict the specificity of MRG1 mAb. 721.221 parental B cellsstably transfected with CEACAM1 (green), CEACAM5 (red), CEACAM6(purple), CEACAM8 (blue) or mock (black), were subjected to FACSanalysis using the different anti-human CEACAM antibodies: MRG1 mAb(FIG. 1A) and Kat4c mAb (FIG. 1B).

FIG. 2 depicts a dose-dependent inhibition of CEACAM1 homophilicinteractions by the anti-CEACAM1 mAb MRG1. Anti-CEACAM1 mAb was added toeither BW/CEACAM1 (effector cells) or 221/CEACAM1 (target cells) invarious concentrations. Following one hour incubation on ice, thereciprocal cells (221/CEACAM1 or BW/CEACAM1) were added and thesecretion of mouse IL-2 was measured by ELISA. 100% is defined as theactivity in the absence of any antibody. The results of onerepresentative experiment out of four are presented, each performed intriplicates.

FIG. 3 depicts abolishment of CEACAM1-inhibitory function. MRG1 mAb waspre-incubated with target cells (depicted in grey) or with effectorcells (depicted in white). Cells incubated without the addition of themAb are depicted in black. The melanoma lines indicated (526mel, 624melor 09mel) were used as target cells. TIL014 cells were used as effectorcells in an E:T ratio of 10:1. Following one hour incubation on ice, thereciprocal cells were added and co-incubated for 5 hours at 37° C.Target cells were pre-labeled with green fluorescent dye (CFSE) andspecific lysis was determined by Propidium Iodide (PI) co-staining inflow cytometry. Spontaneous death was subtracted. Assay was performed intriplicates.

FIG. 4 depicts blocking of melanoma invasion by MRG1 mAbs. Melanomacells (08mel or 09mel) were pre-incubated in the absence or presence of1 μg/ml MRG1 mAb and then tested by Matrigel invasion assays. Invasionwas allowed for 24 hours and the amount of invading cells was quantifiedwith standardized XTT.

FIG. 5 depicts blocking of net proliferation of melanoma cells by MRG1mAbs. 526me1 melanoma cells were incubated with the indicated doses (0.5μg, 1 μg or 3 μg) of MRG1 mAbs and proliferation was monitored 2 days or5 days following treatment.

FIGS. 6A-B depict inhibition of human tumor growth in vivo in SCID miceby systemic injections of MRG1 as compared to PBS. Experiments wereperformed in two setups as follows: FIG. 6A: simultaneous injections ofthe antibody (0.5 mg/mouse intraperitoneally) and inoculation of cancercells (5,000,000 cells subcutaneously); FIG. 6B: treatment of tumorsgenerated in SCID mice (tumor volume of 75 mm³) by injections of MRG1antibody (as indicated above).

FIG. 7 depicts enhanced efficacy in inhibition of tumor growth by acombination of MRG1 with intravenous administration of human reactiveTIL as compared to intravenous TIL only.

FIG. 8 depicts the superior effect of MRG1 mAb over previously describedanti-CEACAM1 monoclonal antibodies, as well as commercially availablerabbit polyclonal antibody targeting human CEACAM1 (DAKO, GlostrupDenmark), as determined by functional blocking assay. Variousanti-CEACAM1 antibodies were tested for blocking of CEACAM1 activity, asreported by mIL-2 secretion. 100% was defined as activity in the absenceof any antibody. The results of one representative experiment out ofthree are presented, each performed in triplicates.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to antiCEACAM1 monoclonal antibody and hybridoma cells producing same as wellas methods of using the antibody in immunomodulation and cancertreatment.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The present inventor has produced through laborious experimentation andscreening a monoclonal antibody selective for CEACAM1. This antibody wasshown to be superior to other anti CEACAM1 monoclonal antibodies asdemonstrated by functional blocking assays.

As is illustrated herein below, the MRG1 antibody produced according tothe present teachings, is selective to CEACAM1 and does not cross reactwith other members of the CEACAM family (i.e., CEACAM 5, 6 and 8, seeExample 2). The antibody inhibits CEACAM1 homophilic interactions, asdetermined by co-incubation of immune effector cells and target melanomacells and assaying IL-2 secretion and cell lysis (see Example 3). Inaddition the antibody was shown effective in inhibiting melanoma cellsinvasion and proliferation. Finally, in vivo administration of theantibody either alone or in combination with reactive lymphocytes wasshown effective in inhibiting growth of melanoma tumors. Altogether, thepresent teachings suggest that the MRG1 antibody, fragments andderivatives can be used as an effective tool for immunomodulation andcancer treatment.

Thus according to an aspect of the invention there is provided ahybridoma cell which has been deposited at the American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va., 20110-2209,United States of America, under ATCC Accession Number PTA-9974, on Apr.29, 2009.

According to a further aspect of the invention there is provided anisolated antibody or antibody fragment comprising an antigen recognitiondomain having the CDR segments and orientation of the antibody producedfrom the hybridoma cell, described above.

The antibody of the present teachings is capable of binding CEACAM1 witha minimal affinity of 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ M.

As used herein the term “CEACAM1” refers to the protein product of theCEACAM1 gene e.g., NP_(—)001020083.1, NP_(—)001703.2.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, and Fvthat are capable of binding to macrophages. According to an exemplaryembodiment the antibody is a monoclonal antibody such as termed herein,MRG1. Functional antibody fragments are defined as follows: (1) Fab, thefragment which contains a monovalent antigen-binding fragment of anantibody molecule, can be produced by digestion of whole antibody withthe enzyme papain to yield an intact light chain and a portion of oneheavy chain; (2) Fab′, the fragment of an antibody molecule that can beobtained by treating whole antibody with pepsin, followed by reduction,to yield an intact light chain and a portion of the heavy chain; twoFab′ fragments are obtained per antibody molecule; (3) (Fab′)2, thefragment of the antibody that can be obtained by treating whole antibodywith the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimerof two Fab′ fragments held together by two disulfide bonds; (4) Fv,defined as a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (5) Single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain and the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule.

As indicated above, the antibody of the present invention has the samecomplementarity determining regions (CDR) orientation as that of theantibody produced by hybridoma cell, having the deposit details asdescribed above. That is CDR1, CDR2, CDR3 are placed in the sameorientation on V_(H) and V_(L) chains.

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by [Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)]. According to some embodiments of thepresent invention, the CDRs can be implemented in any form of anantibody such as by the use of recombinant DNA technology.

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues form acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10,: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

According to some embodiments of the invention, the antibody is attachedto a cytotoxic moiety.

According to some embodiments of the invention, the antibody is attachedto an identifiable moiety.

The identifiable moiety can be a member of a binding pair, which isidentifiable via its interaction with an additional member of thebinding pair and a label which is directly visualized. In one example,the member of the binding pair is an antigen which is identified by acorresponding labeled antibody. In one example, the label is afluorescent protein or an enzyme producing a colorimetric reaction.

The following Table 1 provides examples of sequences of identifiablemoieties.

TABLE 1 Amino Acid Nucleic Acid sequence sequence (Genebank (GenebankIdentifiable Moiety Accession No.) Accession No.) Green Fluorescentprotein AAL33912 AF435427 Alkaline phosphatase AAK73766 AY042185Peroxidase NP_568674 NM_124071 Histidine tag AAK09208 AF329457 Myc tagAF329457 AF329457 Biotin lygase tag NP_561589 NC_003366 orangefluorescent protein AAL33917 AF435432 Beta galactosidase NM_125776NM_125776 Fluorescein isothiocyanate AAF22695 AF098239 StreptavidinS11540 S11540

The cytotoxic or therapeutic moiety can be, for example, a cytotoxicmoiety, a toxic moiety, a cytokine moiety, a bi-specific antibodymoiety, a cytotoxin, a chemokine, a chemotherapy, a pro-apoptotic,interferon, a radioactive moiety, or combinations thereof, examples ofwhich are provided infra.

The following Table 2 provides examples of sequences of therapeuticmoieties.

TABLE 2 Amino acid Nucleic acid sequence sequence (GenBank (GenBankTherapeutic moiety Accession No.) Accession No.) [i Pseudomonas [lexotoxin ABU63124 EU090068 Diphtheria toxin AAV70486 AY820132.1interleukin 2 CAA00227 A02159 CD3 P07766 X03884 CD16 NP_000560.5NM_000569.6 interleukin 4 NP_000580.1 NM_000589.2 HLA-A2 P01892 K02883interleukin 10 P22301 M57627 Ricin toxin EEF27734 EQ975183

It will be appreciated that such fusions can be effected using chemicalconjugation or by recombinant DNA technology.

The antibody of the present invention can decrease the inhibitoryCEACAM1 homophilic (or homotypic) or heterotypic interactions to therebyaugment the activity of lymphocytes. CEACAM1 homophilic interactionsoccur through the N-domain. Several amino acids are crucial for thisinteraction, including R43, Q44, D64 and R82. The interaction causesphosphorylation of a cytoplasmic tyrosine residue that recruits SHP-1phosphatase. This initiates an inhibitory cascade within thelymphocytes, which targets proximal mediators, such as ZAP70.

Thus, the antibody of the present invention can be used to block CEACAM1on either or both immune effector cells (CEACAM1 expressing lymphocytese.g., tumor infiltrating cells, T cells or NK cells) and target cells(e.g., CEACAM1 expressing pathological cells such as cancer cells).Examples of cancer cells which are candidates for this therapy include,but are not limited to, melanoma, lung, thyroid, breast, colon,prostate, hepatic, bladder, renal, cervical, pancreatic, leukemia,lymphoma, myeloid, ovarian, uterus, sarcoma, biliary, or endometrialcells.

The present invention also contemplates isolated antibodies or antibodyfragments-that compete for binding to CEACAM1 with the antibodiesproduced by the above-described hybridoma cell. Those antibodies may behumanized, xenogeneic, or chimeric antibodies (as described in detailabove) being suitable for e.g. therapeutic applications. An antibodyfragment of the antibody can be, for example, a single chain Fvfragment, an F(ab′) fragment, an F(ab) fragment, and an F (ab′)2fragment. Thus, according to a further aspect of the invention there isprovided a method of rendering a CEACAM1 expressing tumor cellsusceptible to immunomodulation. The method comprising contacting theCEACAM1 expressing tumor cell (e.g., melanoma, lung, thyroid, breast,colon, prostate, hepatic, bladder, renal, cervical, pancreatic,leukemia, lymphoma, myeloid, ovarian, uterus, sarcoma, biliary orendometrial cell) with the antibody or antibody fragment describedabove, thereby rendering the CEACAM1 expressing tumor cell susceptibleto immunomodulation.

As used herein “immunomodulation” refers to lymphocyte dependentimmunomodulation (e.g., by NK cells or tumor infiltrating lymphocytes).

Additionally or alternatively, the present invention also envisages amethod of immunomodulation (e.g., inhibiting CEACAM1 homotypic orheterotypic protein-protein interaction), by contacting aCEACAM1-expressing lymphocyte with the antibody or antibody fragmentdescribed herein.

The methods of the present teachings can be effected in-vitro, ex-vivo(e.g., used in T cell based adoptive immunotherapy) or in-vivo.

As mentioned, antibodies of some embodiments of the invention can haveanti cancer activity which is independent from its immunomodulatoryactivity described above.

Thus, the present teachings further provide for a method of inhibitingmigration or proliferation of a CEACAM1 expressing tumor cell, themethod comprising contacting the CEACAM1 expressing tumor cell with theantibody or antibody fragment described herein, thereby inhibitingmigration or proliferation of a CEACAM1 expressing tumor cell.

As used herein “inhibiting” refers to at least 5% , 10%, 20%, 30%, 40%,50%, 60%, 70%, 80% 100% inhibition in proliferation or migration whichcan be assayed using methods which are well known in the art (seeexamples section below).

Antibodies of the present invention can be effectively used for thetreatment of cancer.

Thus according to a further aspect there is provided a method oftreating cancer, the method comprising administering to a subject inneed thereof a therapeutically effective amount of the antibody orantibody fragment described herein, thereby treating the cancer in thesubject. Examples of cancer which can be diagnosed or treated accordingto the present teachings include, but are not limited to, melanoma,sarcoma, lung cancer, cancer of the thyroid, breast cancer, coloncancer, prostate cancer, hepatic cancer, bladder cancer, renal cancer,cervical cancer, pancreatic cancer, leukemia, lymphoma, myeloid cellrelated cancer, ovarian cancer, uterus cancer, biliary cancer orendometrial cancer.

According to a specific embodiment of the present invention, the canceris melanoma.

The term “treating” refers to inhibiting, preventing or arresting thedevelopment of a pathology (disease, disorder or condition) and/orcausing the reduction, remission, or regression of a pathology. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a pathology, and similarly,various methodologies and assays may be used to assess the reduction,remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease,disorder or condition from occurring in a subject who may be at risk forthe disease, but has not yet been diagnosed as having the disease.

As used herein, the term “subject” includes mammals, preferably humanbeings at any age which suffer from the pathology. Preferably, this termencompasses individuals who are at risk to develop the pathology.

In order to enhance treatment (e.g. cancer treatment), lymphocytes suchas T cells (e.g. Tumor Infiltrating Lymphocytes) or NK cells may beadministered to the subject prior to, concomitantly with or followingadministration of the antibody or antibody fragment of the presentinvention. Accordingly, lymphocytes may be obtained from the subject(e.g. from the peripheral blood or from the tumor of same) or from adonor (an allogeneic or a syngeneic lymphocyte donor), treated byex-vivo expansion methods as to obtained viable lymphocytes [e.g. bygrowth on irradiated feeder layer supplemented with IL-2, as previouslydescribed in Besser M J et al., Clin Cancer Res (Epub ahead of print)2010 May 1 and in Besser M J et al., Journal of Immunotherapy (Epubahead of print) 2009 Apr 1, fully incorporated herein by reference] andadministered to the subject.

It will be appreciated that the subject may be treated by any otheranti-cancer treatment (e.g. chemotherapy, radiation therapy, etc.) priorto administration of the antibody or antibody fragment or prior toadministration of the lymphocytes.

The antibody of the present invention can be administered to an organismper se, or in a pharmaceutical composition where it is mixed withsuitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the antibody accountablefor the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, inrtaperitoneal, intranasal, orintraocular injections.

Conventional approaches for drug delivery to the central nervous system(CNS) include: neurosurgical strategies (e.g., intracerebral injectionor intracerebroventricular infusion); molecular manipulation of theagent (e.g., production of a chimeric fusion protein that comprises atransport peptide that has an affinity for an endothelial cell surfacemolecule in combination with an agent that is itself incapable ofcrossing the BBB) in an attempt to exploit one of the endogenoustransport pathways of the BBB; pharmacological strategies designed toincrease the lipid solubility of an agent (e.g., conjugation ofwater-soluble agents to lipid or cholesterol carriers); and thetransitory disruption of the integrity of the BBB by hyperosmoticdisruption (resulting from the infusion of a mannitol solution into thecarotid artery or the use of a biologically active agent such as anangiotensin peptide). However, each of these strategies has limitations,such as the inherent risks associated with an invasive surgicalprocedure, a size limitation imposed by a limitation inherent in theendogenous transport systems, potentially undesirable biological sideeffects associated with the systemic administration of a chimericmolecule comprised of a carrier motif that could be active outside ofthe CNS, and the possible risk of brain damage within regions of thebrain where the BBB is disrupted, which renders it a suboptimal deliverymethod.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

The term “tissue” refers to part of an organism consisting of anaggregate of cells having a similar structure and/or a common function.Examples include, but are not limited to, brain tissue, retina, skintissue, hepatic tissue, pancreatic tissue, bone, cartilage, connectivetissue, blood tissue, muscle tissue, cardiac tissue brain tissue,vascular tissue, renal tissue, pulmonary tissue, gonadal tissue,hematopoietic tissue.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuosinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of a disorder (e.g., cancer) or prolong the survival of thesubject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideantibody levels of the active ingredient are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Therapeutic efficacy can be further validated in correlative animalmodels which are well known in the art. Human xenografts inimmunodeficient mice. Depending on the severity and responsiveness ofthe condition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

Aside from therapeutic applications, antibodies of the present inventioncan also be used in diagnostic applications.

Thus, according to a further aspect there is provided a method fordiagnosing a cancer in a subject in need thereof, the method comprisingcontacting a biological sample derived from the subject (in-vivo orex-vivo) with the antibody or antibody fragment described herein,wherein a complex formation beyond a predetermined threshold isindicative of the cancer in the subject. According to some embodiments,cells of the cancer are characterized by over expression of CEACAM1 ascompared to unaffected cells.

As mentioned, the method of the invention is effected under conditionssufficient to form an immunocomplex; such conditions (e.g., appropriateconcentrations, buffers, temperatures, reaction times) as well asmethods to optimize such conditions are known to those skilled in theart, and examples are disclosed herein. As used herein the phrase“immunocomplex” refers to a complex which comprises the antibody of theinvention and the CEACAM1.

Determining a presence or level of the immunocomplex of the inventionmay be direct or by detecting an identifiable (detectable) moiety whichmay be attached to the antibody.

The level of the immunocomplex in the tested cell (e.g., a cell of asubject in need thereof) is compared to a predetermined threshold. Itwill be appreciated that the antibody of the present invention can alsobe used to measure the amount of serum soluble CEACAM1. Regardless, thethreshold may be determined based on a known reference level and/or alevel in a control cell or serum. The control cell can be obtained froma control, healthy subject (e.g., a subject not suffering from thecancer) or from the same subject prior to disease initiation orfollowing treatment. According to some embodiments of the invention, thecontrol subject is of the same species e.g. human, preferably matchedwith the same age, weight, sex etc. as the subject in need thereof.

As used herein the term “diagnosing” refers to determining presence orabsence of a pathology, classifying a pathology or a symptom,determining a severity of the pathology, monitoring pathologyprogression, forecasting an outcome of a pathology and/or prospects ofrecovery.

To facilitate diagnosis, the above teachings can be combined with othermethods of diagnosing cancer which are well known in the art include butare not limited to imaging, molecular tests and surgical biopsies.

Once the diagnosis is established the subject is informed of thediagnosis and suitable treatments may be initiated.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

The word “exemplary” is used herein to mean “serving as an example,instance or illustration”. Any embodiment described as “exemplary” isnot necessarily to be construed as preferred or advantageous over otherembodiments and/or to exclude the incorporation of features from otherembodiments.

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments”. Any particularembodiment of the invention may include a plurality of “optional”features unless such features conflict.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate some embodiments of the invention in anon limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Generation of Monoclonal Antibodies

Generation of MRG1 monoclonal antibodies

A monoclonal antibody that effectively blocks the CEACAM1 homophilicinteractions in vitro at nanomolar concentrations was generated.Briefly, mice were immunized 3 times, at 2 week intervals, with 5micrograms of recombinant human CEACAM1 (entire protein, commerciallyavailable from R&D Systems). Splenocytes were harvested and fused withSP2/0 cells, to generate a hybridoma library.

The hybridoma producing the CEACAM1-blocking antibody (MRG1 mAb) wasre-cloned several times to yield a stable clone.

Other monoclonal antibodies

Kat4c mAb and rabbit polyclonal anti-CEACAM were purchased from DAKO(Glostrup, Denmark).

Example 2 Specificity of the Anti CEACAM1 mAb

MATERIALS AND EXPERIMENTAL PROCEDURES

Generation of CEACAM expressing cells

CEACAM-negative 721.221 human cells (parental B cells) were stablytransfected with CEACAM1, CEACAM5, CEACAM6 or CEACAM8 by electroporationand selection with G418.

The murine thymoma BW parental cells (cells that lack TCR alpha and betachains, yet retain full secretion machinery of IL-2) were transfectedwith a chimeric molecule comprising the extracellular portion of humanCEACAM1 fused to the transmembrane and cytosolic tail of murine zetachain. Transfection was performed by electroporation and selection withG418.

Antibody screening by FACS

Hybridomas were screened for CEACAM1 binding activity by flow cytometryas follows:

(a) 50,000 transfected CEACAM cells were placed in 96-U shaped wells.

(b) The cells were washed with cold FACS buffer (PBS, BSA 0.5%, Azide0.05%).

(c) The cells were incubated with the staining mAb (MRG1 or Kat4c): 0.1micrograms of mAb per 100 microliters, for 30 minutes, on ice.

(d) The cells were centrifuged, supernatants were removed and the cellswere resuspended in 100 microliter FITC-conjugated goat anti mouseantibodies (Jackson Immunoresearch) at a dilution of 1:200.

(e) After 30 minute incubation (on ice in dark conditions), the cellswere centrifuged, washed and re-suspended in FACS buffer.

(f) Cells were analyzed using a FACScalibur and CellQuest software.

Results

As 721.221 parental cells do not express any of the CEACAM proteins,these cells were stably transfected with CEACAM1, CEACAM5, CEACAM6 orCEACAM8 in order to test the specificity of the CEACAM1 monoclonalantibodies (mAbs). The hybridomas were then screened for CEACAM1 bindingactivity by flow cytometry. As shown in FIG. 1A, the MRG1 mAb generatedaccording to the present teachings is specific to human CEACAM1. It hasan insignificant cross-reactivity to CEACAM5 and no binding to CEACAM6or CEACAM8. FIG. 1B shows that all transfectants expressed CEACAMmolecules, with CEACAM1 being the lowest, which emphasizes thespecificity pattern of MRG1.

Example 3 The mAb is Capable of Inhibiting CEACAM1 Homophilic Binding

Materials and Experimental Procedures

Antibody screening by ELISA

CEACAM1 blocking activity was tested using a BW functional system. TheBW functional system comprises a mouse cell line (BW) stably transfectedwith a chimeric molecule comprising the extracellular domain of humanCEACAM1 fused to mouse zeta chain (BW/CEACAM1-zeta, see Example 2,above). Co-incubation of the BW/CEACAM1-zeta cells with otherCEACAM1-positive cells resulted in the secretion of measurableconcentrations of mouse IL-2.

Thus, BW/CEACAM1-zeta (effector cells) or 221/CEACAM1 (target cells)were each pre-incubated separately with 10-40 ng/ml MRG1 mAb. Followingone hour incubation on ice, the reciprocal cells (221/CEACAM1 orBW/CEACAM1) were added and the secretion of mouse IL-2 was measured bysandwich ELISA (R&D systems).

Cytotoxicity assay

Cytotoxicity assays testing the killing of various melanoma lines bytumor infiltrating lymphocytes was performed in the presence or absenceof 1 μg/ml MRG1 mAb. CEACAM1^(High) 526mel1, 624mel and CEACAM1^(dim)09mel melanoma cells were used as target cells. TIL014 cells were usedas effector cells at an E:T ratio of 10:1. Following one hour incubationwith the MRG1 mAb on ice, the reciprocal cells were added andco-incubated for 5 hours at 37° C. Target cells were pre-labeled with agreen fluorescent dye (CFSE) and specific lysis was determined byPropidium Iodide (PI) co-staining in flow cytometry. Spontaneous deathwas subtracted.

Results

The capability of the purified MRG1 mAb to inhibit CEACAM1 homophilicbinding was verified. As shown in FIG. 2, the purified mAb MRG1 showed adose-dependant inhibition of CEACAM1 homophilic binding. At aconcentration of 10 ng/ml, the mAb efficiently reduced CEACAM1interactions, effectively reaching a plateau at a concentration of 20ng/ml. Importantly, the two experimental settings i.e. the addition ofMRG1 mAb to the effector cells, BW/CEACAM1-zeta, or to the target cells,221/CEACAM1, showed similar results (secretion of the mouse IL-2 waseffectively blocked).

The blocking effect of MRG1 mAb was further demonstrated in cytotoxicityassays. As shown if FIG. 3, killing of the CEACAM1^(High) 526mel and624mel cells was enhanced by incubation of the antibody with effectorcells (but not on target cells). The killing of the CEACAM1^(dim) 09melcells was unaffected by the presence of MRG1 mAb (FIG. 3).

Example 4 Anti CEACAM1 mAb Inhibits Cancer Cell Migration andProliferation

Materials and Experimental Procedures

Invasion assay

The blocking effect of the antibodies was tested in an invasion assay.Briefly, melanoma cells (08mel or 09mel) were pre-incubated in thepresence or absence of 1 μg/ml MRG1 mAb and then tested by Matrigelinvasion assays. Invasion was allowed for 24 hours and the amount ofinvading cells was quantified with standardized XTT.

Net proliferation assay

CEACAM1^(High) 526mel cells were seeded on day 0 in 48-well plates(2,500 cells per well). On seeding, MRG1 was added in 3 differentconcentrations (0.5, 1, or 3 μg/ml), or not added at all. Total viablecells were counted 2 days or 5 days after seeding. Proliferation wasdetermined with standardized XTT and by direct cell counting.

Results

As shown in FIG. 4, MRG1 blocked the invasion of CEACAM1-positive 08melcells (CEACAM1 expression level was medium, i.e. median fluorescenceintensity of CEACAM1 expression was 50) and had little or no effect onCEACAM1dim 09mel cells (CEACAM1 expression level was low, i.e. medianfluorescence intensity of CEACAM1 expression was 15).

MRG1 was also tested in net proliferation assays. A dose-dependentinhibition in net proliferation of 526mel cells was observed (FIG. 5).Following 5 days of treatment, proliferation was reduced by more than60% (with 3 μg MRG1 mAb).

Example 5 MRG1 Inhibits Cancer Cell Growth in Animal Experimental Models

Materials and Experimental Procedures

Melanoma xenograft models

5×10⁶ CEACAM1⁺ human melanoma cells were injected subcutaneously to theflank of 7 week old SCID-NOD mice. Tumor masses formed in 100% of themice within 14-17 days and continued to grow. Tumor dimensions weremonitored non-invasively with a caliper 3 times a week and volumeapproximation was calculated as (d1×d2×d3/2).

Administration of MRG1 was performed by injection of 0.5 mg antibodydiluted in 0.5 ml sterile PBS intraperitoneally. Injection of PBS servedas control.

Administration of reactive human anti-melanoma lymphocytes was performedby intravenous injection into the tail vein of 20×10⁶ cells diluted in200 μl of sterile PBS.

Results

In line with the blocking functions demonstrated above, administrationof MRG1 antibody inhibited tumor growth. This effect was evident whenthe antibody was administered at the time of tumor cell inoculation(FIG. 6A, “Prevention setup”) or after a measurable tumor mass wasalready formed (FIG. 6B, “Treatment setup”). These effects were evidentafter 4 injections within 8 days, followed by non-invasive monitoring(see arrows in FIG. 6). It should be noted that this effect wasindependent of any immunomodulating effect, as SCID-NOD mice areimmunodeficient.

Simulation of anti-melanoma immune response was performed by a singleintravenous injection of reactive human anti-melanoma lymphocytes, whichinhibited tumor growth (FIG. 7). This effect was significantly enhancedby intraperitoneal MRG1 injections once a week.

Example 6 MRG1 is Superior to Previously Described Anti-CEACAM1Antibodies

Materials and Experimental Procedures

Antibody screening by ELISA

CEACAM1 blocking activity was tested using a BW functional system asdescribed in detail in Example 3, hereinabove.

100,000 BW/CEACAM1-zeta cells were pre-incubated with 15 ng/ml MRG1 mAb,2600 ng/ml Kat4c mAb or 600ng/ml rabbit polyclonal anti-CEACAM antibody.Following one hour incubation on ice, 50,000 721.221/CEACAM1 cells wereadded and the secretion of mouse IL-2 was measured by sandwich ELISA(R&D Systems).

RESULTS

As depicted in Example 3, hereinabove, the inventors demonstrated anearly complete blocking of CEACAM1 activity using 15 ng/ml MRG1 mAb. Incontrast, the anti-CEACAM1 monoclonal antibody Kat4c was able to yield aminor blocking effect only when 200-fold higher concentrations weretested and the polyclonal rabbit anti-CEACAM antibody yielded a similarinhibitory effect with 40-fold higher concentration (2600 ng/ml and600ng/ml, respectively, FIG. 8).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A hybridoma cell which has been deposited underATCC Accession Number PTA-9974.
 2. An isolated antibody or antibodyfragment that recognizes CEACAM1, and that comprises the same heavychain variable region CDR1, CDR2 and CDR3 sequences, the same lightchain variable region CDR1, CDR2 and CDR3 sequences, and the sameorientation as the CDRs of the antibody produced from a hybridoma cellwhich has been deposited under ATCC Accession Number PTA-9974.
 3. Theisolated antibody or antibody fragment of claim 2 attached to acytotoxic moiety.
 4. The isolated antibody or antibody fragment of claim3, wherein said cytotoxic moiety comprises a cytotoxin, a chemokine, achemotherapy, a pro-apoptotic, an interferon, a radioactive moiety, orcombinations thereof.
 5. The isolated antibody or antibody fragment ofclaim 2 attached to an identifiable moiety.
 6. A method ofimmunomodulation, the method comprising contacting a CEACAM1-expressinglymphocyte with the antibody or antibody fragment of claim
 2. 7. Amethod of inhibiting migration or proliferation of a CEACAM1 expressingtumor cell, the method comprising contacting the CEACAM1 expressingtumor cell with the antibody or antibody fragment of claim 2, therebyinhibiting migration or proliferation of a CEACAM1 expressing tumorcell.
 8. A method for determining the expression of CEACAM1, the methodcomprising contacting a biological sample with the antibody or antibodyfragment of claim 2, and measuring the level of immune complexformation.
 9. A method of treating a CEACAM1-expressing cancer, themethod comprising administering to a subject in need thereof atherapeutically effective amount of the antibody or antibody fragment ofclaim 2, thereby treating the cancer in the subject.
 10. The method ofclaim 9, further comprising administering to the subject lymphocytes.11. The method of claim 10, wherein said lymphocytes comprise T cells orNK cells.
 12. A method of inhibiting CEACAM1 homotypic or heterotypicprotein-protein interaction, the method comprising contacting aCEACAM1-expressing lymphocyte with the antibody or antibody fragment ofclaim 2, thereby inhibiting CEACAM1 homotypic or heterotypicprotein-protein interaction.
 13. The method of claim 6, wherein saidCEACAM1-expressing lymphocyte is a Tumor Infiltrating Lymphocyte or NKcell.
 14. The method of claim 6, wherein said CEACAM1-expressinglymphocyte is a cytotoxic T cell.
 15. The method of claim 7, whereinsaid tumor cell comprises a melanoma tumor cell.
 16. A pharmaceuticalcomposition comprising as an active ingredient the antibody or antibodyfragment of claim
 2. 17. The isolated antibody or antibody fragmentaccording to claim 2 wherein the antibody or antibody fragment isselective to humand CEACAM1.
 18. The isolated antibody or antibodyfragment according to claim 2 produced from the hybridoma cell which hasbeen deposited under ATCC Accession Number PTA-9974.